Development of new dye-metal agarose-coated alumina matrix and elution strategy for purification of alcohol dehydrogenase

A high-density matrix was prepared by coating an alumina particle with agarose using an emulsion technique. Iminodiacetic acid and Cibacron Blue 3GA were immobilized onto this matrix. Charging this matrix with zinc created a useful chromatography matrix for purification of yeast alcohol dehydrogenase. The elution strategy was then investigated to obtain a high recovery of this enzyme and a high purification factor. One-step elution using 4 mM EDTA containing 0.5 M NaCl resulted in 66% enzyme recovery and a purification factor of 4.7. Two-step elution using imidazole containing NaCl resulted in a higher purification factor. The first-step elution using 5 mM imidazole containing 1 M NaCl released most contaminant proteins. The second-step elution using 150 mM imidazole containing 1.5 M NaCl resulted in high-performance purification with a purification factor of 6.5 and an enzyme recovery of 40.7%. Equilibration of the matrix with imidazole prior to sample application increased the purification factor and the enzyme recovery to 8.4 and 76.8%, respectively.